A High‐Throughput Mass‐Spectrometry‐Based Assay for Identifying the Biochemical Functions of Putative Glycosidases

Abstract

The most common glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position of potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a mass spectrometry-based glycosidase assay amenable to high-throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high-throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved by observing a correct mass-loss between a potential sugar substrate and its corresponding product using electrospray ionization mass spectrometry (ESI-MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly-expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus.

Directed Evolution: Methodologies and Applications

“…Automation can be applied at different stages in directed evolution to reduce cost and time, thus improving throughput. Robotic liquid handling approaches based on pipet tips, , pins, inkjet, and acoustic deposition , have been applied to accelerate assay setup and sample preparation. Moreover, during screening/selection, automated introduction of samples into a mass spectrometer enables substantial improvements in analytic throughput.…”

Automation in cell and gene therapy manufacturing: from past to future

“…Examples include CyBio® (Analytik Jena), RoboLector (M2P Labs) (Kensy et al 2009), Cello™ robot (Sartorius), Biomek® 4000 (Beckman), Freedom EVO (Tecan), STAR system (Hamilton), SimCell™. (Lindgren et al 2009; Warr et al 2009) These systems have been used successfully for cell culture applications (US Food and Drug Administration 2017), cell line development (Deng et al 2011; Lindgren et al 2009), cell characterisation or even for nanoscale assay development (Mosquito® - TTP Labtech) (Goadsby et al 2017). However none of these systems are fully automated platforms capable of supporting a bioprocess from start to finish.…”

Scope and limitations of carbohydrate hydrolysis for de novo glycan sequencing using a hydrogen peroxide/metallopeptide-based glycosidase mimetic

“…In order to screen a wide spectrum of carbohydrate linkages, an expanded library of methyl glycosides—designed earlier to interrogate proteinaceous glycosidase function [23, 24]—was used to set up an activity screen of the CuGGH metallopeptide-based artificial glycosidase in a high-throughput format. A high throughput robotic liquid handling system was used to dispense a small volume of liquid (as low as 1.2 nl) laterally within columns in a 384-well plate with a change of tips in between each transfer step to avoid cross-contamination.…”

“…A high throughput robotic liquid handling system was used to dispense a small volume of liquid (as low as 1.2 nl) laterally within columns in a 384-well plate with a change of tips in between each transfer step to avoid cross-contamination. In addition to the previously reported ten common monosaccharide substrates[23], three new, commercially available, methyl glycoside substrates (methyl-α-L-rhamnopyranoside, methyl- N -acetyl-α-D-glucosaminide and methyl-β-L-arabinopyranoside) were added to the existing library for an expanded activity screen. All catalytic components (hydrogen peroxide, sodium ascorbate and CuGGH metallopeptide) were substituted with deionized water for the negative control.…”